Noguchi A, Furuno T, Kawaura C, Nakanishi M
Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
FEBS Lett. 1998 Aug 14;433(1-2):169-73. doi: 10.1016/s0014-5793(98)00837-0.
By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl-3beta-carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein-conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS-7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.
通过共聚焦激光扫描显微镜（CLSM），我们研究了阳离子脂质体与参与阳离子脂质体介导的基因转染的内体膜之间的膜融合。反义寡核苷酸由阳离子脂质体与阳离子胆固醇衍生物胆甾醇-3β-羧酰胺基乙二甲基胺（I）进行转运。阳离子脂质体由衍生物I和二油酰磷脂酰乙醇胺（DOPE）的混合物制成。通过CLSM研究了荧光素偶联的反义寡核苷酸（硫代磷酸酯）的细胞内分布。图像显示，带有衍生物I的脂质体将反义寡核苷酸优先转运到靶细胞（NIH3T3、COS-7和HeLa细胞）的细胞核中。然而，尼日利亚菌素能够消除跨内体膜的pH梯度，尽管在靶细胞的细胞质中发现了脂质体/DNA复合物，但它完全阻断了它们的转染。这与用渥曼青霉素（一种内吞作用抑制剂）处理的靶细胞的荧光图像形成了鲜明对比。结果表明，对于由带有阳离子胆固醇衍生物的阳离子脂质体介导的基因转染，至少有两个步骤是有效的。一个是脂质体/DNA复合物被内吞进入靶细胞，另一个是在内体中从复合物中去除反义寡核苷酸（质粒DNA）。后一步骤优先于pH 5.0左右阳离子脂质体与内体膜之间的膜融合。
